This project is a continuation of our efforts to elucidate the basis of receptor-activated presynaptic modulation. Our hypothesis is that inhibitory modulators act by increasing K conductance. Utilizing rat cortical synaptosomes that are prelabeled with either 86Rb or [3H] acetylcholine, (ACh) we have shown that when transmitter release is evoked with either KC1 or veratridine, the addition of inhibitory presynaptic modulators to the medium results in an increased efflux and a decrease in ACh release. Since these results support our hypothesis we now plan to enlarge this synaptosomal study by 1) determining the specific K channel that is implicated in presynaptic modulation using relatively specific K channel blockers. 2) investigating presynaptic modulators that increase transmitter release, and 3) utilizing pertussis-toxin treated preparations, determine if G-proteins are always intermediaries. A second aim is to identify and characterize Substance B, a regulatory factor we discovered in nervous tissue that has no effect on evoked ACh release but reverses inhibitory modulated release, regardless of whether the modulating agent is muscarinic, adrenergic, purinergic or opioid. When the identity of this novel regulator has been established, we will determine its subcellular distribution, its biochemical and electrophysiological mechanism of action, its effect on transmitters other than acetylcholine, and if behavioral effects are observed when Substance B is administered intracerebrally.